Functional identification of protein phosphatase 1-binding consensus residues in NBCe1-B

Protein phosphatase 1 (PP1) is involved in various signal transduction mechanisms as an extensive regulator. The PP1 catalytic subunit (PP1c) recognizes and binds to PP1-binding consensus residues (FxxR/KxR/K) in NBCe1-B. Consequently, we focused on identifying the function of the PP1-binding consensus residue, ⁹²²FMDRLK⁹²⁷ , in NBCe1-B. Using site-directed mutagenesis and co-immunoprecipitation assays, we revealed that in cases where the residues were substituted (F922A, R925A, and K927A) or deleted (deletion of amino acids 922–927), NBCe1-B mutants inhibited PP1 binding to NBCe1-B. Additionally, by recording the intracellular pH, we found that PP1-binding consensus residues in NBCe1-B were not only critical for NBCe1-B activity, but also relevant to its surface expression level. Therefore, we reported that NBCe1-B, as a substrate of PP1, contains these residues in the C-terminal region and that the direct interaction between NBCe1-B and PP1 is functionally critical in controlling the regulation of the HCO₃⁻ transport. These results suggested that like IRBIT, PP1 was another novel regulator of HCO₃⁻ secretion in several types of epithelia.

Ahcyl2 upregulates NBCe1-B via multiple serine residues of the PEST domain-mediated association

Inositol-1,4,5-triphosphate [IP3] receptors binding protein released with IP3 (IRBIT) was previously reported as an activator of NBCe1-B. Recent studies have characterized IRBIT homologue S-Adenosylhomocysteine hydrolase-like 2 (AHCYL2). AHCYL2 is highly homologous to IRBIT (88%) and heteromerizes with IRBIT. The two important domains in the N-terminus of AHCYL2 are a PEST domain and a coiled-coil domain which are highly comparable to those in IRBIT. Therefore, in this study, we tried to identify the role of those domains in mouse AHCYL2 (Ahcyl2), and we succeeded in identifying PEST domain of Ahcyl2 as a regulation region for NBCe1-B activity. Site directed mutagenesis and coimmunoprecipitation assay showed that NBCe1-B binds to the N-terminal Ahcyl2-PEST domain, and its binding is determined by the phosphorylation of 4 critical serine residues (Ser151, Ser154, Ser157, and Ser160) in Ahcyl2 PEST domain. Also we revealed that 4 critical serine residues in Ahcyl2 PEST domain are indispensable for the activation of NBCe1-B using measurement of intracellular pH experiment. Thus, these results suggested that the NBCe1-B is interacted with 4 critical serine residues in Ahcyl2 PEST domain, which play an important role in intracellular pH regulation through NBCe1-B.